Method of treating autoimmune diseases

ABSTRACT

A method of treating an autoimmune disease comprising administering to the subject a treatment effective amount of a histone hyperacetylating agent, or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 10/151,481, filed May 20, 2002, which is acontinuation-in-part of U.S. application Ser. No. 09/718,195, filed Nov.21, 2000, and claims priority from PCT Application No. PCT/US01/43871,filed Nov. 19, 2001, the disclosures of which are incorporated herein byreference in their entireties.

STATEMENT OF FEDERAL SUPPORT

This invention was made possible with government support under grantnumbers R01 AR39501 from the National Institute of Health. The UnitedStates government may have certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of autoimmunediseases such as systemic lupus erythematosus or rheumatoid arthritis.

BACKGROUND OF THE INVENTION

The hallmark of the aberrant cellular immune response in systemic lupuserythematosus (SLE) is T cell dysfunction (A. K. Dayal and G. M. Kammer,Arthritis Rheum. 39, 23 (1996); D. A. Horwitz, et al., in Dubois' LupusErythematosus., D. J. Wallace and B. H. Hahn, Eds. (Williams & Wilkins,Baltimore, 1997), chap. 10). An imbalance exists between exaggeratedhelper function and deficient cytotoxic/suppressor activity thatpromotes inappropriate B cell overproduction of immunoglobulins (Ig).The resulting polyclonal hypergammaglobulinemia is comprised of naturalantibodies and pathogenic autoantibodies, including anti-native DNA.Formation of complement-fixing immune complexes in situ or theirdeposition on vascular endothelium, such as the renal glomerulus,initiates a chronic inflammatory response that leads to irreparableparenchymal damage, ultimately resulting in end-organ failure (R. P.Kimberly, in Arthritis and Allied Conditions: A Textbook ofRheumatology, W. J. Koopman, Ed. (Williams & Wilkins, Baltimore, 1997)chap. 27). Moreover, T cell dysfunctions predispose to recurrent, oftenlife-threatening infections. See, A. G. Iliopoulos and G. C. Tsokos,Sem.Arthritis Rheum. 25, 318 (1996); C. A. Hunter and S. L. Reiner,Curr.Opin.Immunol. 12, 413 (2000).

Two principal defects of T cell function in SLE are augmented expressionof cell surface receptors and altered production of cytokines. CD40ligand (CD154) expression is significantly increased and prolonged onboth CD4⁺ helper (Th) and CD8⁺ cytotoxic/suppressor (Tc) subpopulations(M. Koshy, et al., J.Clin.Invest. 98, 826 (1996); A. Desai-Mehta, et al,J.Clin.Invest. 97, 2063 (1996)). This prolonged over-expression may bepathophysiologically significant, for binding of CD154 on Th cells toCD40 on B cells promotes B cell activation and may drive the polyclonalhypergammaglobulinemia. Moreover, Th2 cells over-produce IL-10 whereasTh1 cells under-produce IFN-γ. Heightened levels of IL-10 may profoundlymodify the cellular immune response by (a) downregulating both IFN-γ andIL-2 production by Th1 cells; (b) inhibiting IL-12 generation anddown-regulating expression of IL-12 receptors on Th1 cells; (c)up-regulating bcl-2 expression and preventing apoptosis of activated Tcells; and, (d) promoting B cell growth, differentiation andautoantibody production. By contrast, deficient IFN-γ may significantlyhinder cellular immunity in SLE by both impairing Tc-dependentcytotoxicity and altering antigen-presentation (B. S. Handwerger, etal., in Lupus: Molecular and Cellular Pathogenesis, G. M. Kammer and G.C. Tsokos, Eds. (Humana Press, Totowa, N.J., 1999), chap. 21).

Histone deacetylases (HDACs) are enzymes that deacetylate specificlysine residues of histone amino-terminal tail domains and certainnon-histone substrates. Current evidence implicates the deacetylases intranscriptional repression (T. Kouzarides, Curr.Opin.Genet.Dev. 9, 40(1999); W. D. Cress and E. Seto, J.Cell.Physiol. 184, 1 (2000)).Complexed with Sin3 and Mi2 transcriptional co-repressor proteins,HDAC/Sin3 and HDAC/Mi2 associate with other DNA-binding proteins, suchas Ikaros (W. D. Cress and E. Seto, J.Cell.Physiol. 184, 1 (2000); J.Kim et al., Immunity 10, 345 (1999)). These deacetylase complexes appearto limit the accessibility of transcription factors to the promoter byclosely juxtaposing the nucleosome to DNA. Of the eight human HDACsdiscovered (W. D. Cress and E. Seto, J.Cell.Physiol. 184, 1 (2000)), todate only HDACs1-3 have been identified in T cells (F. Dangond et al.,Biochem.Biophys.Res.Comm. 242, 648 (1998)). During T cell activation,HDAC/Mi2 complexes are recruited to regions of the heterochromatin byIkaros and modulate gene expression (J. Kim et al., Immunity 10, 345(1999); Koipally, J., et al. EMBO J 18, 3090 (1999)). Trichostatin A, anHDAC inhibitor (M. Yoshida, et al., J.Biol.Chem. 265, 17174 (1990); S.Finnin et al., Nature 401, 188 (1999)), blocks deacetylase activity andshifts the equilibrium toward histone acetylation. By acetylatinghistones, chromatin is remodeled, promoting access of DNA-bindingtranscription factors and the transcriptional machinery topromoter/enhancer regions (W. D. Cress and E. Seto, J. Cell.Physiol.184, 1 (2000); R. D. Kornberg and Y. Lorch, Curr.Opin.Gen.Dev. 9, 148(1999)). Acetylation may mediate positive or negative regulatory eventsthat depend upon the particular gene (Z. W. Sun and M. Hampsey, Genetics152, 921 (1999)). Thus, promoter regions that are ordinarily silencedcan then be derepressed whereas those that are expressed can berepressed. However, the use of histone deacetylase inhibitors or otherhistone hyperacetylating agents in the treatment of autoimmune diseasessuch as SLE has not heretofore been suggested or disclosed.

While several treatments for autoimmune diseases such as SLE andrheumatoid arthritis have been developed, none are entirelysatisfactory. Hence, there remains a need for new ways to treatautoimmune diseases.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of treating anautoimmune disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ahistone hyperacetylating agent, or a pharmaceutically acceptable saltthereof.

A second aspect of the present invention is a method of treatingSystemic Lupus Erythematosus in a subject in need thereof, comprisingadministering to that subject a therapeutically effective amount of ahistone hyperacetylating agent, or a pharmaceutically acceptable saltthereof.

A still further aspect of the present invention is the use of an activeagent as described above for the preparation of a medicament for thetreatment of a disorder as described above.

Still further aspects of the present invention are methods of treatingan autoimmune disease in a subject in vivo.

Another aspect of the present invention includes methods a treating anautoimmune disease in a subject comprising the administration of apharmaceutical formulation to the subject.

Yet another aspect of the present invention comprises administering acompound to treat an autoimmune disease.

The present invention is explained in greater detail in thespecification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the down-regulation of CD154 transcript levels bytrichostatin A (TSA). Increasing concentrations of TSA (0-1000 ng/ml)progressively inhibit expression of CD154 mRNA relative to expression ofGAPDH mRNA.

FIG. 1B shows a graphic depiction of a densitometric scan of the gel inFIG. 1A. This graph depicts the percent change of CD154 mRNA expressionwith increasing concentrations of TSA over 24 hr. GAPDH mRNA expressionis stable and unchanged in the presence of TSA.

FIG. 1C illustrates CD154 transcript levels in T cells incubated in theabsence or presence of 1000 ng/ml TSA over 18 hr. T cells were thenstimulated with 20 ng/ml PMA+0.5 μM IO for intervals to 24 hr. CD154mRNA expression relative to GAPDH mRNA expression is shown.

FIG. 1D shows a graphic depiction of the percent change in CD154 mRNAexpression over time in the absence (filled circles) or presence (opencircles) of TSA.

FIG. 1E demonstrates flow cytometric analysis of CD154 and CD3-εexpression on SLE T cells. T cells were cultured in the absence orpresence of 1000 ng/ml TSA for 18 hr, and subsequently activated with 20ng/ml PMA+0.5 μM IO for 24 hr. The abscissa denotes the number of cellsand ordinate the intensity of cell fluorescence signal. Statisticalanalyses were performed by paired Student's t test or one-way ANOVA.

FIG. 2A depicts the down-regulation of IL-10 levels by TSA. Increasingconcentrations of TSA (0-1000 ng/ml) progressively inhibit expression ofIL-10 mRNA relative to expression of GAPDH mRNA.

FIG. 2B shows a graphic depiction of a densitometric scan of the gel inFIG. 2A. This graph depicts the percent change of IL-10 transcriptexpression with increasing concentrations of TSA over 24 hr.

FIG. 2C shows IL-10 and GAPDH transcripts from T cells of three SLEsubjects. Transcripts from freshly isolated T cells are shown in lanes1, 4, and 7. Transcripts from T cells cultured for 18 hr in the absenceor presence of 1000 ng/ml TSA are shown in lanes 2, 5, 8 and 3, 6, and9, respectively.

FIG. 2D shows a graphic depiction of a densitometric scan of the gel inFIG. 2C. This graph shows the percent change in IL-10 mRNA from SLE Tcells cultured in the absence or presence of 1000 ng/ml TSA.

FIG. 2E illustrates the inhibition of IL-10 secretion by increasingconcentrations of TSA over 24 hr.

FIG. 2F depicts the percent change of IL-10 production over time.Statistical analysis was performed by paired Student's t test.

FIG. 3A shows the up-regulation of IFN-γ transcript by TSA. T cells wereincubated in the absence or presence of 1000 ng/ml TSA over 18 hr. Tcells were then stimulated with 20 ng/ml PMA +0.5 μM 10 for intervals to24 hr. IFN-γ mRNA expression relative to GAPDH mRNA expression is shown.

FIG. 3B demonstrates a graphic depiction of a densitometric scan of thegel in FIG. 3A. This graphs depicts the fold increase of IFN-γ mRNA incells cultured in the absence (filled circles) or presence (opencircles) of 1000 ng/ml TSA during intervals to 24 hr.

FIG. 3C illustrates IFN-γ protein levels from T cells cultured in theabsence or presence of 1000 ng/ml TSA for 24 hr, and then stimulatedwith 20 ng/ml PMA+0.5 μM 10 for 24 hr. The graph shows the fold increaseof IFN-γ protein secretion. Statistical analyses were performed bypaired Student's t test or one-way ANOVA.

FIG. 4A is a bar graph showing that real time PCR was used to quantitatethe amount of TGF-β1 mRNA relative to GAPDH mRNA in cells treated withincreasing amounts of TSA.

FIG. 4B illustrates a comparison of the effect of TSA in normal vs. SLEpatients.

FIG. 5 depicts a Western blot analysis of acetylated histone H3 and H4protein in SLE PBMCs treated with TSA or SAHA.

FIGS. 6A-6F demonstrate that TSA induces accumulation of acetylated H3and H4 histones in chromatin associated with the TGF-β1 gene.Specifically, FIG. 6A is a schematic diagram of the promoter regions;FIG. 6B illustrates chromatin fragments from vehicle or TSA treated SLEPBMCs that were immunoprecipitated with normal IgG, anti-acetylated H3or anti-acetylated H4 antibodies, and wherein PCR primer sets are forthe regions indicated in lane A; FIGS. 6C and 6D are the quantitation ofCHIP from SLE (C) or normal controls (D); FIG. 6E demonstrates that TSAdoes not change acetylated state of G6PD gene in SLE PBMCs; and FIG. 6Fis the quantitation of FIG. 6E.

FIGS. 7A-7C depict Chromatin conformation of the TGF-β1 gene promoteranalyzed by DNase I sensitivity assay.

FIGS. 8A-8D illustrate that TSA and SAHA downregulate IFN-γ transcriptand protein levels. FIG. 8A shows that TSA decreases the levels of IFN-γmRNA relative to GAPDH mRNA in MRL/lpr splenocytes; FIG. 8B shows thatTSA and SAHA prevent induction of IFN-γ mRNA by Con A; FIG. 8C indicatesa fold change of IFN-γ mRNA shown in FIG. 8B; and FIG. 8D is a graphdepicting the amount of IFN-γ protein secretion, wherein the barrepresents the mean±SEM of three independent experiments.

FIGS. 9A-9E demonstrate that TSA and SAHA downregulates IL-12 p35 andp40 transcript and IL-12 p40 protein levels. FIG. 9E depicts the amountof IL-12 p40 protein secretion.

FIGS. 10A-10D show that TSA and SAHA downregulate IL-6 transcript andprotein levels.

FIGS. 11A-11D show that TSA and SAHA downregulate IL-10 transcript andprotein levels.

FIG. 12 illustrates a Western blot analysis of acetylated histone H3 andH4 protein in MRL/lpr splenocytes treated with TSA or SAHA.

FIGS. 13A-13B examine whether histone deacetylase inhibitors couldsuppress inducible nitric oxide synthetase. FIG. 13A illustrates thesemiquantitative RT-PCR of iNOS in MRL/lpr splenocytes, and FIG. 13Billustrates the production of nitric oxide from MRL/lpr mesangial cells,respectively.

FIGS. 14A-14B are bar graphs illustrating the decrease in spleen weight(A) and proteinuria (B) in TSA.

FIG. 15 is a graph showing the renal score for mice treated with vehicleor TSA.

FIG. 16 shows two graphs demonstrating the autoantibody production inmice treated with vehicle or TSA where the sera levels of anti-dsDNA andanti-GBM were measured at 14 and 19 weeks of age, respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, etc.

The phrase “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

A “therapeutically effective” amount as used herein is an amount of ahistone hyperacetylating agent that is sufficient to treat autoimmunediseases in a subject. The therapeutically effective amount will varywith the age and physical condition of the patient, the severity of thetreatment, the duration of the treatment, the nature of any concurrenttreatment, the pharmaceutically acceptable carrier used and like factorswithin the knowledge and expertise of those skilled in the art.

Active compounds of the present invention may optionally be administeredin conjunction with other compounds useful in the treatment of theautoimmune disease such as SLE. The other compounds may optionally beadministered concurrently. As used herein, the word “concurrently” meanssufficiently close in time to produce a combined effect (that is,concurrently may be simultaneously, or it may be two or more eventsoccurring within a short time period before or after each other).

As used herein, the administration of two or more compounds“concurrently” or “in combination” means that the two compounds areadministered closely enough in time that the presence of one alters thebiological effects of the other. The two compounds may be administeredsimultaneously or sequentially. Simultaneous administration may becarried out by mixing the compounds prior to administration, or byadministering the compounds at the same point in time but at differentanatomic sites or using different routes of administration.

Autoimmune diseases with which the present invention is concernedinclude, but are not limited to, rheumatoid arthritis, systemic lupuserythematosus, alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, autoimmune Addison's disease, autoimmune hemolytic anemia,autoimmune hepatitis, Behcet's Disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunedysfunction syndrome, chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoblobulinemia, fibromyalgia, fibromyositis, Goodpasture syndrome,graft versus host disease, Grave's disease, Guillain-Barre syndrome,Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathicthrombocytopenia purpura, IgA nephropathy, insulin dependent diabetes,juvenile arthritis, lichen planus, lupus, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis, dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, Raynaud's Phenomenon, Reiter's syndrome,rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Mansyndrome, Takayasu Arteritis, temporal arteritis, giant cell arteritis,ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. A particularly preferred application of the presentinvention is in the treatment of systemic lupus erythematosus (SLE).

The present invention is primarily concerned with the treatment of humansubjects, but the invention may also be carried out on animal subjects,particularly mammalian subjects such as mice, rats, dogs, cats,livestock and horses for veterinary purposes, and in vitro for drugscreening and drug development purposes. In addition, the presentinvention may be used to treat animal subjects that are models of anautoimmune disease for drug screening and development purposes. Aparticular example of such a model is the mouse NZB/NZW F1 model of SLE.

Active Compounds.

Active compounds used to carry out the present invention are, ingeneral, histone hyperacetylating agents, such as histone deacetylaseinhibitors. Numerous such compounds are known. See, e.g., P. Dulski,Histone Deacetylase as Target for Antiprotozoal Agents, PCT ApplicationWO 97/11366 (Mar. 27, 1997). Examples of such compounds include, but arenot limited to:

Trichostatin and its analogues, such as: Trichostatin A (TSA); andTrichostatin C (Koghe et al. 1998. Biochem. Pharmacol. 56:1359-1364).

Peptides, such as: Oxamflati [(2E)-5-[3-[(phenylsufonyl) aminophenyl1]-pent-2-en-4-ynohydroxamic acid (Kim et al., Oncogene,18:2461-2470 (1999)); Trapoxin A (TPX)—Cyclic Tetrapeptide(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy-decanoyl))(Kijima et al., J. Biol. Chem. 268, 22429-22435 (1993)); FR901228,Depsipeptide (Nakajima et al., Ex.Cell Res. 241, 126-133 (1998));FR225497, Cyclic Tetrapeptide (H. Mori et al., PCT Application WO00/08048 (Feb. 17, 2000)); Apicidin, Cyclic Tetrapeptide[cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93, 13143-13147(1996)); Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin IIa, andApicidin IIb (P. Dulski et al., PCT Application WO 97/11366); HC-Toxin,Cyclic Tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950 (1995));WF27082, Cyclic Tetrapeptide ( PCT Application WO 98/48825); andchlamydocin (Bosch et al., supra).

Hydroxamic Acid-Based Hybrid Polar Compounds (HPCs), such as:Salicylihydroxamic Acid (SBHA) (Andrews et al., International J.Parasitology 30, 761-768 (2000)); Suberoylanilide Hydroxamic Acid (SAHA)(Richon et al., Proc. Natl. Acad. Sci. USA 95, 3003-3007 (1998));Azelaic Bishydroxamic Acid (ABHA) (Andrews et al., supra);Azelaic-1-Hydroxamate-9-Anilide (AAHA) (Qiu et al., Mol. Biol. Cell 11,2069-2083 (2000)); M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Riconet al., supra); 6-(3-Chlorophenylureido)carpoic Hydroxamic Acid(3-Cl-UCHA) (Richon et al., supra); MW2796 (Andrews et al., supra); andMW2996 (Andrews et al., supra). Note that analogs not effective as HDACInhibitors are: Hexamethylene bisacetamide (HBMA) (Richon et al. 1998,PNAS, 95:3003-3007); and Diethylbix(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA) (Richon etal. 1998, PNAS, 95:3003-3007).

Short Chain Fatty Acid (SCFA) Compounds, such as: Sodium Butyrate(Cousens et al., J. Biol. Chem. 254, 1716-1723 (1979)); Isovalerate(McBain et al., Biochem. Pharm. 53:1357-1368 (1997)); Valerate (McBainet al., supra); 4-Phenylbutyrate (4-PBA) (Lea and Tulsyan, AnticancerResearch, 15, 879-873 (1995)); Phenylbutyrate (PB) (Wang et al., CancerResearch, 59, 2766-2799 (1999)); Propionate (McBain et al., supra);Butrymide (Lea and Tulsyan, supra); Isobutyramide (Lea and Tulsyan,supra); Phenylacetate (Lea and Tulsyan, supra); 3-Bromopropionate (Leaand Tulsyan, supra); and Tributyrin (Guan et al., Cancer Research, 60,749-755 (2000)).

Benzamide derivatives, such as: MS-27-275[N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA 96,4592-4597 (1999)); and 3′-amino derivative of MS-27-275 (Saito et al.,supra).

Other inhibitors, such as: Depudecin [its analogues (mono-MTM-depudecinand depudecin-bisether) do not inhibit HDAC) (Kwon et al. 1998. PNAS95:3356-3361); and Scriptaid (Su et al. 2000 Cancer Research,60:3137-3142). Additionally, other inhibitors, such as: isoquinolinswhich would include scriptaid and nullscipt.

The compounds may include the general formula:

wherein each of R₁ and R₂ is, substituted or unsubstituted, hydrogen,aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,t-butyl, aryloxy, arylalkoyloxy, phenyl or pyridine group; and whereinR₂ may be attached by a linker such as an amido moiety, —O—, —S—, —NH—or —CH₂—; and wherein n is an integer from 3 to 8.

The active compounds disclosed can, as noted above, be prepared in theform of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of such salts are (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine, and (c) salts derived from bases, such as ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium, and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

Compounds for Concurrent Administration.

The active compound histone hyperacetylating agents described herein maybe administered concurrently with other active compounds known for thetreatment of autoimmune diseases (such as systemic lupus erythematosus).Examples of such other active compounds include, but are not limited to:(i) corticosteroids such as prednisolone sodium phosphate, such asPediapred®; methylprednisolone, such as Medrol®; prednisone, such asDeltasone® or Orasone®; and dexamethasone, such as Decadron® Tablets;(ii) steroids such as lynestrenol—a progestagen; desogestrel—aprogestagen; ethylestrenol—an anabolic steroid; and tibolone—a weakprogestational, anabolic, androgenic steroid (H. A. Verheul et al. Clin.Immunol. Immunopathol. 38:198-208 (1986)); and exogenousDHEA—dehydroepiandrosterone—(T. Suzuki et al. Clin. Exp. Immunol.99:251-255 (1995)); and (iii) other compounds such as hydroxchloroquinesulfate, such as Plaquenil®; H1-A (isolated from Cordyceps sinensis) (L.Y. Yang, et al. J. Lab Clin. Med. 134:492-500 (1999)); sulfasalazine(a.k.a. Salazosulfapyridine) (E. Delaporte et al. Ann. Dermatol.Venereol. 124:151-156 (1997)); anti-ICAM-1—murine antiintercellularadhesion molecule-1 (R. L. Brey et al. Lupus 6:645-651 (1997));MX-68—upolyglutamable antifolate (M. Mihara et al. Int. Arch. AllergyImmunol. 13:454-459 (1997)); FK506—(K. Yamamoto et al. Immunology69:222-227 (1990)); AS101—organotellurium compound—(J. Alcocer-Varela etal. Clin. Exp. Immunol. 77:319-323 (1989));HWA-131-(3-(3,5-ditert.butyl-4-hydroxyphenyl)-7H-thiazolo(3,2-b)(1,2,4)triaz in-7-one) (R. R. Bartlett et al. Drugs Exp. Clin. Res.15:521-526 (1989)); and Auranofin—Oral gold compound—(K. Dalziel et al.Br. J. Dermatol. 115:211-216 (1986)).

The foregoing may be administered in the same formulation and/or by thesame route of administration, or by a different formulation and/ordifferent route of administration, as the active agent histonehyperacetylating agents described herein, in their conventional dosagesor dosages which can be determined from the conventional dosages.

Pharmaceutical Formulations.

The active compounds described above may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the active compound (including thephysiologically acceptable salts thereof) is typically admixed with,inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be incorporated inthe formulations of the invention, which may be prepared by any of thewell known techniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous,etc.), topical (e.g., both skin and mucosal surfaces, including airwaysurfaces) and transdermal administration, although the most suitableroute in any given case will depend on the nature and severity of thecondition being treated and on the nature of the particular activecompound which is being used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an active compoundas described above, or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mactive ingredient.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the compounds disclosedherein or salts thereof, may be lyophilized to produce a lyophilizatewhich may be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

In addition to active compounds or their salts, the pharmaceuticalcompositions may contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the compositions may contain microbialpreservatives. Useful microbial preservatives include methylparaben,propylparaben, and benzyl alcohol. The microbial preservative istypically employed when the formulation is placed in a vial designed formultidose use. Of course, as indicated, the pharmaceutical compositionsof the present invention may be lyophilized using techniques well knownin the art.

Dosage and Routes of Administration.

As noted above, the present invention provides pharmaceuticalformulations comprising the active compounds (including thepharmaceutically acceptable salts thereof), in pharmaceuticallyacceptable carriers for oral, rectal, topical, buccal, parenteral,intramuscular, intradermal, or intravenous, and transdermaladministration.

The therapeutically effective dosage of any specific compound, the useof which is in the scope of present invention, will vary from compoundto compound and patient to patient, and will depend upon factors such asthe age, weight, gender and condition of the patient and the route ofdelivery. As a general proposition, a dosage from about 0.01 or 0.1 toabout 50, 100 or 500 mg/kg will have therapeutic efficacy, with allweights being calculated based upon the weight of the active compound,including the cases where a salt is employed. Toxicity concerns at thehigher level may restrict intravenous dosages to a lower level such asup to about 10 mg/kg, with all weights being calculated based upon theweight of the active base, including the cases where a salt is employed.A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg maybe employed for intramuscular injection. Preferred dosages are 0.01mg/kg to 50 mg/kg of the compound for intravenous or oraladministration. The duration of the treatment is usually once per dayfor a period of two to, three weeks or until the condition isessentially controlled. Lower doses given less frequently can be usedprophylactically to prevent or reduce the incidence of recurrence of thedisorder, or the severity of symptoms. For example, the trichostatinanalog SAHA is being given in phase I clinical trials for cancer by anintravenous route.

Screening Assays.

The present invention also provides screening assays for identifyingcompounds useful, or potentially useful, in the treatment of autoimmunediseases such as SLE. Such assays may be carried out in accordance withknown techniques, such as the formats described in P. Dulski, PCTApplication WO97/11366 (March 27, 1997).

One method of screening compounds for activity in treating an autoimmunedisease, comprises: contacting a histone deacetylase, or an extractcontaining histone deacetylase with (i) a known amount of a labeledcompound that interacts with a histone deacetylase; and (ii) a knowndilution of a test compound or natural product extract; and thendetermining the inhibition of interaction of said labeled compound withsaid histone deacetylase induced by said test compound, where theinhibition of interaction of said labeled compound with said histonedeacetylase indicates said compound or extract is a candidate for thetreatment of an autoimmune disease.

The histone deacetylase is preferably a mammalian (e.g., mouse, rat,rabbit) histone deacetylase, and is most preferably a human histonedeacetylase. The labeled compound may be any of the active agentsdescribed above, labeled with a suitable detectable group such astritium. In general, the labeled compound will be one which binds tohistone deacetylase or is a substrate of histone deacetylase. The testcompound may be of any source, such as an oligomer or a non-oligomerfrom a combinatorial library, or a rationally synthesized candidatecompound. Extracts may be obtained from any suitable source, such asplant extracts obtained through techniques known in traditional, folk orherbal medicine. The determining step may be carried out qualitativelyor quantitatively by any suitable means, such as by Scatchard analysiswith a series of serial dilutions of the test compound or extract.

In another embodiment, a method of screening compounds for activity intreating an autoimmune disease such as SLE comprises: contacting anintact host cell in vivo or in vitro with a test compound or a naturalproduct extract; and then determining the level of histone acetylationin said cell, wherein elevated levels of histone acetylation indicatessaid compound or extract is a candidate for the treatment of anautoimmune disease.

Where the contacting step is carried out in vivo (e.g., as in the courseof a clinical trial) the compound is administered to a suitable subjectcarrying the cell by any of the same techniques described above foradministering active agents, and the cell (or collection of cells)subsequently collected from the subject for use in the determining step.The cell (or subject) is preferably mammalian (e.g., a mouse, rat orrabbit cell) and in one particularly preferred embodiment is human.Lymphocytes are particularly preferred cells. The subject may be oneafflicted with an autoimmune disease such as SLE (including models ofsuch a disease), or may be a normal (or unafflicted) subject. Elevatedlevels may be determined by comparison to an untreated, control subjector cell, by comparison to levels found in the same subject or cell orcell population prior to treatment, etc. Assays for histone levels maybe carried out by any suitable technique, with histone level assaysbeing known to those skilled in the art.

The examples, which follow, are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof. In thefollowing examples, hr means hour; min means minute; TSA meansTrichostatin A; SLE means systemic lupus erythematosus; RT-PCR meansreverse transcriptase polymerase chain reaction; 10 means ionomycin, PMAmeans phorbol myristate acetate, ml means milliliter; ng means nanogram;and all temperatures, unless otherwise indicated, are in degreesCelsius.

EXAMPLE 1 Down-Regulation of CD154 Transcript and Protein Levels by TSA

Because SLE T cells are often activated (D. T. Y. Yu et al., J.Exp.Med.151, 91 (1980).), the up-regulation of CD154 and IL-10 anddown-regulation of IFN-γ may reflect skewed gene expression due toenhanced recruitment of HDACs to the promoters of these genes. Theresulting disequilibrium of acetylation might be expected to alter thechromatin structure of the promoters (R. D. Kornberg and Y. Lorch,Curr.Opin.Gen.Dev. 9, 148 (1999)), thereby activating previouslysilenced genes while repressing expressed genes. To determine if TSA candown-regulate CD154 transcript expression, T cells from eight SLEsubjects were treated with increasing concentrations of TSA over 18 hr.

T cells were cultured in the absence or presence of increasingconcentrations of TSA for 18 hr in 5% CO₂ at 37° C. RNA was isolated,cDNAs were prepared, and RT-PCR was performed as previously detailed (D.Laxminarayana, et al., J. Clin. Invest. 92, 2207 (1993)). The primersused were: (SEQ ID NO: 1) CD154: 5′-GAATCCTCAAATTGCGGCAC-3′ and (SEQ IDNO: 2) 5′-CAGAAGGTGACTTGGCATAG-3′; (SEQ ID NO: 3) GAPDH:5′-GGTGAAGGTCGGAGTCAACG-3′ and (SEQ ID NO: 4)5′-CAAAGTTGTCATGGATGACC-3′; (SEQ ID NO: 5) IL-10:5′-TTGCCTGGTCCTCCTGACTG-3′ and (SEQ ID NO: 6)5′-GATGTCTGGGTCTTGGTTCT-3′; (SEQ ID NO: 7) IFN-γ:5′-ATGAAATATACAAGTTATATCTTGGCTTT-3′ and (SEQ ID NO: 8)′-GATGCTCTTCGACCTCGAAACAGCAT-3′.

The reaction mixtures were subjected to 30 cycles of denaturation (94°C., 1 min) and annealing for 1 min at 53° C. (CD154), 40° C. (GAPDH) and55° C. (IL-10 and IFN-γ). Extension was for 2 min at 72° C. with a finalextension of 7 min at 72° C. using a DNA thermal cycler (Perkin-Elmer).

FIGS. 1A and 1B demonstrate that TSA maximally inhibits CD154 transcriptby 60%, but does not modify GAPDH MRNA expression. When SLE T cells wereactivated with phorbol myristate acetate (PMA) and ionomycin (10), CD154mRNA content increased 100%, peaked at 3 hr, and waned thereafter (FIG.1C and 1D). Under these conditions, however, GAPDH MRNA remained stable,demonstrating that cellular activation also does not modify theexpression of this gene. By contrast, when T cells were preincubatedwith TSA for 18 hr and then activated by PMA+10 over intervals to 24 hr,up-regulation of CD 154 transcript was significantly reduced throughoutthe entire time course compared to cells not exposed to TSA (FIG. 1C and1D; P<0.001). Thus, in SLE T cells TSA significantly down-regulatesCD154 transcript expression.

In agreement with previous work (M. Koshy, et al., J.Clin.Invest. 98,826 (1996); A. Desai-Mehta, et al., J.Clin.Invest. 97, 2063 (1996)), wefind that an increased proportion of SLE T cells express cell-surfaceCD154 compared to normal and disease controls. To determine ifTSA-dependent down-regulation of CD154 mRNA reduces surface expressionof CD154, SLE T cells were treated for 18 hr with TSA and the proportionof CD154⁺ cells quantified by flow cytometry (E. Hagiwara, et al.,Arthritis Rheum. 39, 379 (1996)). Compared with untreated cells, TSA didnot effect any significant reduction of cell-surface CD154⁺ cells over24 hr (FIG. 1E). However, activation of SLE T cells with PMA+10 over 24hr induced a new population of CD154⁺ cells that was completelyinhibited when cells were pretreated with TSA prior to activation (FIG.1E; P=0.005). By contrast, CD3-ε expression remained stable under thesevarying conditions, indicating that TSA's effect on CD 154 surfaceexpression is not generalized (FIG. 1E). T cells were stained withsaturating concentrations of monoclonal FITC-anti-CD3 and PE anti-CD154antibodies (Caltag Labs, Burlingame, Calif.) for 30 min at 4° C., andthe proportion of cells expressing CD3-ε and CD154 was quantified. Insum, these experiments reveal that TSA down-regulates both CD154 mRNAand protein expression, but not GAPDH mRNA or CD3-E expression, in SLE Tcells.

EXAMPLE 2 Down-Regulation of IL-10 Transcript and Protein Levels by TSA

T cells from SLE subjects produce markedly increased amounts of IL-10resulting in high serum levels of the cytokine (B. S. Handwerger, etal., in Lupus: Molecular and Cellular Pathogenesis, G. M. Kammer and G.C. Tsokos, Eds. (Humana Press, Totowa, N.J., 1999) chap. 21; E.Hagiwara, et al., Arthritis Rheum. 39, 379 (1996)). To determine whetherTSA could down-regulate IL-10, a dose-response analysis was performed.Like CD154, increasing concentrations of TSA progressively inhibitedIL-10 transcript expression (FIG. 2A and 2B). In fact, based onsensitive reverse transcriptase-polymerase chain reaction (RT-PCR)analyses, no detectable IL-10 mRNA was identified at TSA concentrationsof 700-800 ng/ml. By comparison, increasing concentrations of TSA didnot modify GAPDH transcript expression (FIG. 2A and 2B). As shown inFIG. 2C, IL-10 transcripts were present in freshly isolated T cells (0hr; lanes 1, 4, 7) and remained stable relative to GAPDH transcriptsafter culturing cells for 18 hr (lanes 2, 5, 8). However, when SLE Tcells were cultured in the presence of TSA for 18 hr, no detectableIL-10 transcripts were identified (FIG. 2C, lanes 3, 6, 9). When IL-10transcripts from all eight SLE subjects were quantified relative toGAPDH transcripts, TSA inhibited expression of IL-10 mRNA by 71% (FIG.2D; P=0.029). Treatment of T cells from eight SLE subjects over 18 hrwith increasing concentrations of TSA resulted in a dose-dependentinhibition of IL-10 protein production that was maximal at 300 ng/ml ofthe inhibitor (FIG. 2E). IL-10 and IFN-γ protein production werequantified by ELISA (R & D Systems, Minneapolis, Minn.). Within 6 hr,TSA inhibited IL-10 production by 90%; at 24 hr, there was completeinhibition of IL-10 synthesis (FIG. 2F). Thus, like CD154, TSA was ableto block expression of IL-10 transcript, abolishing IL-10 production bySLE T cells.

EXAMPLE 3 Up-Regulation of IFN-γ Transcript and Protein Levels by TSA

Low production of IFN-γ by SLE T cells may reflect down-regulation ofgene expression (B. S. Handwerger, et al., in Lupus: Molecular andCellular Pathogenesis, G. M. Kammer and G. C. Tsokos, Eds. (HumanaPress, Totowa, N.J., 1999), chap. 21; E. Hagiwara, et al., ArthritisRheum. 39, 379 (1996)). To establish whether TSA can up-regulate IFN-γexpression, SLE T cells were treated for 18 hr in the absence orpresence of TSA. During that time, TSA induced a three-fold increase inIFN-γ transcript compared to untreated cells (FIG. 3A, lanes 1 and 7,and FIG. 3B). When T cells were activated with PMA+IO in the absence ofTSA, peak IFN-γ transcript expression increased 13-fold at 1 hr overbasal levels relative to GAPDH transcript, but waned thereafter. Bycontrast, activation of T cells in the presence of TSA induced a peak37-fold increase in IFN-γ mRNA at 6 hr over untreated cells relative toGAPDH (FIG. 3A and 3B; P=0.031). Thus, TSA up-regulated expression ofIFN-γ transcripts in SLE T cells, yielding both a significantlyincreased and prolonged expression of the transcript.

This strong up-regulation of IFN-γ transcript was reflected insignificantly increased production of IFN-γ protein by 24 hr. In theabsence of stimulation, SLE T cells failed to produce any IFN-γ over 72hr. When T cells were activated with PMA+IO for 24 hr, IFN-γ productionincreased about 24-fold. However, activation of T cells in the presenceof TSA further enhanced IFN-γ output by >12-fold (P=0.011) (FIG. 3C).Taken together, these results demonstrate that TSA rapidly up-regulatesboth IFN-γ transcript and protein production by SLE T cells.

The capacity of TSA to down-regulate cell surface CD154 and IL-10production and to up-regulate IFN-γ synthesis in SLE T cells providesnew evidence in support of the proposition that skewed gene expressionmay be a fundamental mechanism underlying both the cellular and humoralimmune dysregulation in this disease. That TSA was able to modify thisaltered gene expression in vitro also supports the concept that HDACsmay be recruited to the promoter regions of these genes where theyeffect skewed expression. Because the precise mechanism by which histoneacetylation modifies transcription still remains uncertain (T.Kouzarides, Curr.Opin.Genet.Dev. 9, 40 (1999)), it is also unclear howinhibition of HDAC activity by TSA effects down-regulation of CD154 andIL-10 and up-regulation of IFN-γ in SLE T cells. Notwithstanding, thiscapacity of TSA to modulate the expression of these genes appears tohave the salutary effect of normalizing their protein expression invitro. Because it can simultaneously target multiple genes involved inthe immunopathogenesis of lupus, TSA would be an effective therapeuticagent.

In SLE, a chronic inflammatory response progressively destroys organparenchyma, ultimately leading to irreversible end-organ failure such asend-stage renal disease. The immunopathogenesis of this chronicinflammatory process is in part due to the presence of complement-fixingimmune complexes. Formation of pathogenic immune complexes depends onproduction of autoantibodies, such as anti-native DNA, that arise fromdysregulated B cell clones (B. H. Hahn, New Engl. J. Med. 338, 1359(1998)). Therefore, down-regulation of CD154 and IL-10 should eliminateboth the sustained CD154-CD40 interaction as well as high cytokinelevels that drive polyclonal hypergammaglobulinemia and autoantibodyproduction, reducing immune complex formation. Similarly, up-regulationof IFN-y production might be expected to normalize an abnormal cellularimmune response that predisposes to infections.

EXAMPLE 4 Up-Regulation of TGF-β1

Transforming growth factor β (TGF-β) derives from a family ofpleiotropic cytokines that exhibit opposing biologic functions and isknown to inhibit IL-1, IL-6 and TNF-β production by macrophages as wellas IgG and IgM secretion by activated B cells in vitro. Kitamura et al.,“Transforming growth factor-beta 1 is the predominant paracrineinhibitor of macrophage cytokine synthesis produced by glomerularmesangial cells”, J Immunol 156:2964 (1996). Both constitutive andstimulated production of TGF-β by lymphocytes is significantly lower inSLE than controls. Ohtsuka et al., “Decreased production of TGF-beta bylymphocytes from patients with systemic lupus erythematosus” J Immunol160:2539 (1998). This down-regulation of TGF-β may reflect thesuppressive effects of high IL-10 levels on the cytokine's production byNK cells. Addition of TGF-β and IL-2 to SLE PBMCs almost completelyabolishes the increased, spontaneous production of IgG by these cellsand injection of TGF-β c-DNA into MRL/lpr mice decreases autoantibodyproduction and prolongs their life-expectancy.

In contrast, the addition of anti-TGF-β mAb to SLE PBMCs resulted inincreased spontaneous Ig synthesis, suggesting that decreased TGF-βleads to B cell hyperactivity. Del Giudice et al., “Role of transforminggrowth factor beta (TGF beta) in systemic autoimmunity”, Lupus 2:213(1993). This is further supported by the observation that TGF-β knockoutmice reveal striking evidence of autoimmunity that resembles human SLE.Dang et al., “SLE-like autoantibodies and Sjogren's syndrome-likelymphoproliferation in TGF-beta knockout mice” J Immunol 155:3205(1995). The sera of these mice contain elevated levels of predominantlyIgG anti-dsDNA, -Sm and -ribonucleoprotein autoantibodies.

Gene expression of TGF-β1 was tested to determine if the gene expressionis suppressed by deacetylation of histones in SLE. In thisdetermination, SLE PBMCs or isolated T cells were treated with thehistone deacetylase inhibitors (HDIs), TSA or SAHA, for 18 hr. As shownin FIG. 4A, concentrations of TSA to 500 ng/ml stimulated a modestincrease in TGF-β1 mRNA, whereas 1000 ng/ml TSA resulted in an 8-foldinduction of TGF-β1 mRNA in SLE T cells compared to vehicle treatedcells. Subsequently, semi-quantitative PCR was performed to determinethe amounts of TGF-β1 transcripts in SLE and control T cells treatedwith vehicle or TSA (1000 ng/ml). FIG. 4B demonstrates that TSAincreased TGF-β1 mRNA levels in SLE, but not in healthy control T cells.There is no change in GAPDH transcript in normal and SLE T cells aftertreatment of HDIs. Increased TGF-β1 transcript was associated with asubsequent increase in TGF-β1 protein secretion by SLE PBMCs. Whencompared to vehicle treated PBMCs, total TGF-β1 protein in culturesupernatants from SLE PBMCs increased by approximately 30%. In contrast,there was no increase in total TGF-β1 secretion in TSA treated cells innormal healthy controls. Similar results may be obtained by SAHA.

The effect of TSA and SAHA was examined to determine if HDIs induceaccumulation of acetylated histones in SLE PBMCs on the level of histoneacetylation in SLE PBMCs. Following culture of PBMCs in the presence ofvehicle, 1000 ng/ml TSA or 20 μM SAHA for 18 hr, histones were isolated.Western immunoblot analysis showed that the level of acetylated H3 andH4 histones in vehicle-treated cells was low (FIG. 5, lane 1). Bycontrast, incubation with TSA or SAHA induced the accumulation ofacetylated H3 and H4 histones (FIG. 5, compare lane 1 with 2 and 5).Stimulation of SLE PBMCs with PMA and lonomycin alone did not result inincreased accumulation of either acetylated histone H3 or H4 (FIG. 5,lane 3).

The effect of HDAC inhibition on the acetylation of H3 or H4 associatedwith the TGF-β1 gene promoter was examined using the chromatinimmunoprecipitation (ChIP) assay in an attempt to determine if HDIsinduce accumulation of acetylated histones in chromatin associated withthe TGF-β1 promoter in SLE PBMCs but not in healthy normal controls. Thestudy undertaken included immunoprecipitating formaldehyde cross-linked,sonicated chromatin fragments from vehicle- and HDI-treated cells usinganti-acetyl H3 and H4 antibodies, respectively. The immunoprecipitatedDNA released from the bound protein was analyzed by quantitative PCR. Aseries of primer sets encompassing the TGF-β1 gene from −1.4 kbp to +0.6kbp relative to the start site of transcription were created (FIG. 6A).Approximately a 12- and 4-fold enhancement of acetylated histones H3 andH4, respectively, in the. TGF-β1 promoter in SLE was observed (FIGS. 6B& 6C). By contrast, essentially no enhancement of acetylated histones H3and H4 at the TGF-β1 promoter in normal PBMCs treated with TSA was seen(FIG. 6D). The finding that silencing of the TGF-β1 gene is relieved byTSA may suggest that it is mediated by a mechanism leading todeacetylation of histones associated with its promoter in SLE PBMCs, butnot in normal healthy controls.

To determine whether this effect was selective for the TGF-β1 gene inSLE PBMCs, the level of histone acetylation in the glucose-6-phosphatedehydrogenase (G6PD) gene was also examined. As shown in FIGS. 6E & 6F,there was no change in the levels of acetylated histones H3 and H4 atthe G6PD gene in SLE PBMCs.

Because acetylated histones are generally associated withtranscriptionally active chromatin whereas deacetylated histones areoften found in conjunction with an inactive chromatin state, adetermination of whether HDAC inhibition could alter chromatin structureat the TGF-β1 gene locus was performed. Because nuclease susceptibilityis one of the characteristics of active chromatin, a DNase I sensitivityassay was used to examine chromatin conformation of the TGF-β1 gene inSLE PBMCs in the absence or presence of TSA. SLE PBMCs were treated with1000 ng/ml of TSA for 18 hr, a treatment course shown previously toresult in an optimal increase of TGF-β1 mRNA. Equal amounts of purifiednuclei isolated from vehicle- or TSA-treated cells were exposed toincreasing concentrations of DNase I (FIGS. 7A & 7B). Changes in DNasesensitivity were measured by a DNase I-PCR assay using 0.5 μg of DNA. Asshown in the FIG. 7A, the cells treated with only vehicle wererelatively resistant to DNase I digestion, even with use of higherconcentrations of DNase I (0.25 μg/μl). In contrast, cells treated withTSA were digested in the lower concentration (0.025 μg/μl) of DNase I(FIG. 7B; lanes 3-6). TSA treatment resulted in a three-fold increase inDNase I sensitivity (FIG. 7C), suggesting that inhibition of HDACactivity leads to a more open chromatin conformation in the TGF-β1promoter of SLE PBMCs. There was no change in DNase I sensitivity at theG6PD gene promoter in vehicle- or TSA-treated SLE PBMCs (FIGS. 7A & 7B).Thus, it is likely that Trichostatin A increases DNase I sensitivity inthe TGF-β1 gene of SLE PBMCs.

Additionally, because production of TGF-β1 by SLE lymphocytes ismarkedly impaired, (See, Ohtsuka et al., J Immunol 160, 2539-2545(1998)), the hypothesis that HDAC may be associated with transcriptionalrepression of the TGF-β1 gene was tested and resulted in reduced TGF-β1production. Exposure of SLE PBMCs to TSA and SAHA yielded an eight-foldincrease in TGF-β1 transcript and a 30% rise in the synthesis of TGF-β1protein. This enhanced transcriptional activation was temporallyassociated with increased acetylation of both H3 and H4 nucleosomalhistones. Utilizing the ChIP assay, a twelve- and four-fold increase wasdemonstrated in acetylated H3 and H4 histones in the TGF-β1 promoter,respectively. The increase in total acetylated histones was not global;neither GAPDH nor G6PD genes were transcriptionally activated.Importantly, localized acetylation of the TGF-β1 promoter aftertreatment with TSA was not associated with a concomitant increase in SP1binding. This suggests that TSA and SAHA modulate TGF-β1 promoteractivity by a mechanism other than increasing the DNA binding capacityof SP1. Thus, the capacity of HDIs to increase the endogenous synthesisof TGF-β1 raises the possibility that these inhibitors could betherapeutic candidates for the treatment of SLE, atherosclerosis, andosteoporosis in addition to cancers.

EXAMPLE 5 Murine Models of SLE

Animal models provide a powerful tool to study disease mechanisms and totest novel therapeutic agents under well-defined conditions. Severalmice model of lupus have been well characterized. The next few examplesuse MRL/lpr/lpr mice in their studies. The MRL/lpr/lpr mice closelyresemble the human disease of SLE.

MRL/lpr/lpr Mouse Model of Lupus

Murphy and Roths developed the MRL/lpr strain and the congenic MRL/++ in1976. They were derived from LG/J mice crossed with AKR/J, C3HDi, andC57BI/6. By the twelfth generation of inbreeding, MRL/lpr was derived.This mouse strain has a single spontaneous autosomal recessive genemutation (Ipr) of the fas apoptosis gene on chromosome 19. Interactionsof Fas and Fas ligand (FasL) are required for the initiation ofapoptosis in activated B and T lymphocytes under normal immunoregulatoryconditions. Therefore, mice that are homozygous for the lpr mutation(i.e., lpr/lpr) develop massive lymphoproliferation, large quantities ofIgG autoantibodies, and autoimmune disease.

Both male and female MRL/lpr mice develop high serum levels ofimmunoglobulins, monoclonal paraproteins, ANAs, and immune complexes atabout 6 wks of age (4, 5). Males lag behind females by approximately 1month. By 12-16 wks of age, there is serologic evidence of a panoply ofautoantibodies IgM and IgG anti-ssDNA and anti-dsDNA andhypocomplementemia. Other autoantibodies in their repertoire include IgGantibodies that bind chromatin, histone, nucleosomes, nucleobindin(i.e., a DNA-binding protein), cardiolipin, erythrocyte surfaces,thyroglobulin, lymphocyte surfaces, Sm, U1 snRNP, Ro, La, Ku, Su,proteoglycans on endothelial cell membranes, neurons, ribosomal P, RNApolymerase I, Clq, and heat-shock proteins. A substantial portiondevelop IgG3 cryoglobulins, some containing rheumatoid factor activity.They develop clinical signs of arthritis, massive lymphadenopathy, skindisease, severe necrotizing arteritis, and glomerulonephritis (GN) bythe age of 16-24 wks. Fifty percent of mice die from renal failure by 24wk of age. Most MRL/lpr mice develop lymphocytic infiltration ofsalivary glands, pancreas, peripheral muscles and nerves, uvea, andthyroid. In fact, they develop clinical disease of hypothyroidism,abnormal electrical transmission in muscles and nerves (suggestingclinical polymyositis and polyneuritis), learning disabilities,sensorineural hearing loss, and band keratopathy. The thymus isstructurally abnormal in MRL/lpr mice, as it is in all strains thatdevelop spontaneous SLE. Thymic cortical atrophy is severe and medullaryhyperplasia common. The numbers of epithelial cells in the subcapsularand medullary regions are decreased. Total cortical thymocytes aredecreased in number. Thymectomy of newborn MRL/lpr mice preventsdevelopment of lymphoproliferation and autoimmune disease and MRL/lprthymus engrafted into MRU+/+ mice causes lymphoproliferation and earlydeath from autoimmune nephritis.

Polyarthritis occurs in some MRL/lpr mice with prevalence between15-25%. By 14 wks of age, there is synovial cell proliferation withearly subchondral bone destruction and marginal erosion. Acutenecrotizing arteritis, primarily of coronary and renal arteries, isfound in over 50% of MRL/lpr mice. Many develop myocardial infarctions.

MRL/++ Mice

MRL/+/+ mice share over 95% of the genetic material of MRL/lpr mice butdiffer at the lpr locus. MRL/+/+ mice are auto-immune prone and developlate-life lupus. They make anti-DNA, anti-Sm, and rheumatoid factors,but serum levels are lower than those of MRL/lpr mice. Disease inMRL/+/+mice does not show a gender bias, as male and females aresimilarly affected. Most mice develop clinical nephritis with advancingage.

C57BI/6J-lpr (B6/lpr/lpr) Mice (6)

B6/lpr/lpr mice do not develop arthritis, skin disease,glomerulonephrits or arteritis. They do not develop splenomegaly orlymphadenopathy until 8 months of age whereas MRL/lpr mice show lymphoidhyperplasia beginning at 4 months of age. B6/lpr/lpr mice have delayed50% mortality (at 12 months) compared to MRL/lpr mice, which exhibit 50%mortality at 6 months of age.

EXAMPLE 6 Down-Regulation of Th1 and Th2 Cytokine Gene Expression

In determining whether TSA can down-regulate IFN-γ mRNA, splenocytesfrom older, 24-wk MRL/lpr mice were treated with 0 to 500 ng/ml of TSAfor 18 hr. This interval was selected based on our data demonstratingoptimal down-regulation of CD154 and IL-10 transcripts in human SLE Tcells by TSA. Compared to splenocytes from 10-wk old mice that had lowIFN-γ mRNA (FIG. 8B, lane 1), older mice had higher constitutive levelsof IFN-γ transcripts normalized to the housekeeping gene, GAPDH (FIG.8A, lane 1). Although low concentrations of TSA initially induced anincrease in IFN-γ mRNA (FIG. 8A, lane 2), further increase in theconcentration of TSA progressively inhibited IFN-γ mRNA (FIG. 8A, lanes3 & 4). Maximal inhibition of IFN-γ transcript consistently occurred at500 ng/ml of TSA. In contrast, expression of the housekeeping gene,GAPDH, remained stable. These results demonstrate that the heightenedexpression of IFN-γ mRNA in 24 wk old MRL/lpr splenocytes can bedown-regulated by TSA.

To establish whether TSA or SAHA down-regulates Con A-induced IFN-γ mRNAexpression, splenocytes from 10-wk old mice were stimulated with Con A(10 μg/ml) for 18 hr. From previous kinetic studies, IFN-γ mRNA ismaximally expressed when T cells are activated for 18 hr. Con Astimulation consistently up-regulated IFN-γ mRNA content approximately2.5-fold, but did not alter GAPDH mRNA expression (FIGS. 8B & 8C, lanes1 vs. 2). In contrast, when splenocytes were preincubated with TSA orSAHA for 18 hr, up-regulation of IFN-γ transcript was markedly reducedcompared with cells treated with the vehicle alone (FIGS. 8B & 8C, lanes3-6). Dose titration experiments revealed that the optimal dose of SAHAwas 10 μM (data not shown). Thus, both TSA and SAHA inhibit IFN-γ mRNAup-regulation in response to mitogenic stimulation.

The marked inhibition of IFN-γ transcript by these inhibitors promptedus to quantify IFN-γ protein secretion in splenocyte culturesupernatants. Over 72 hr, Con A, but not LPS, stimulated splenocytesfrom younger MRL/lpr mice to secrete a mean SEM) 1,563.2±88.3 μg/ml ofIFN-γ (FIG. 8D). Con A-stimulated splenocytes cultured in the presenceof either SAHA or TSA did not secrete detectable levels of IFN-γ protein(p=0.004, ANOVA FIG. 8D). Thus, inhibition of IFN-γ transcription by TSAand SAHA blocks secretion of IFN-γ protein by splenocytes.

Next, TSA and SAHA were tested to determine if they down-regulateexpression of IL-12 p40 and IL-12 p35 mRNA, and IL-12 p40 protein. It isknown in the art that IL-12 is a 75-kD cytokine comprised of aheterodimer with p35 and p40 subunits that are essential for thedifferentiation of the Th1 subset of CD4⁺ T cells. However, at the levelof transcription, each of these subunits is regulated independently. Itpromotes differentiation of Th₁ CD4⁺ T cells. Administration ofrecombinant IL-12 (rIL-12) to younger MRL/lpr mice accelerates GNwhereas anti-IL-12 mAb inhibits production of anti-dsDNA autoantibody inNZB/W F₁ mice.

In this study, the question of whether TSA down-regulates transcriptionof both IL-12 p35 and p40 subunit mRNA was investigated. Splenocytesfrom 24-wk MRL/lpr mice revealed constitutive expression of both IL-12subunit transcripts (FIG. 9A). However, increasing concentrations of TSAabove 100 ng/ml completely suppressed both transcripts (FIG. 9A, lanes3&4). To address whether TSA and/or SAHA down-regulate LPS- and IFN-Yinduced IL-12 p35 and p40 transcripts, splenocytes from 10-wk oldMRL/lpr mice were incubated with vehicle, TSA or SAHA for 18 hr prior tostimulation. Splenocytes were stimulated with LPS and IFN-γ for 6 or 18hr. FIGS. 9B-D demonstrate that, at 6 hr, there was a 9- and 3.5-foldincrease of IL-12 p40 and p35 transcripts, respectively, when stimulatedwith LPS and IFN-γ following vehicle treatment; at 18 hr, thefold-increase was 6- and 1.5-fold for IL-12 p40 and p35 mRNA,respectively. When splenocytes were preincubated with TSA or SAHA for 18hr and then stimulated with LPS and IFN-γ IL-12 p35 and p40 transcriptswere undetectable (FIGS. 9B-D).

In the absence of stimulation, splenocytes did not secrete detectableIL-12 p40 after 24 hr in culture. When splenocytes were activated withLPS (100 μg/ml) and IFN-γ 100 U/ml) for 24 hr the mean (±SEM) IL-12 p40secretion was 45.3±9.1 pg/ml. However, stimulation of splenocytes in thepresence of TSA or SAHA yielded significantly lower IL-12 p40 secretionafter 24 hr (mean±SEM, TSA=1.5±1.4 pg/ml; SAHA 9.1±1.9 pg/ml; p=0.003 byANOVA) (FIG. 9E). Taken together, these results may demonstrate thattreatment with TSA and SAHA leads to decreased levels of IL-12 p35 andp40 transcripts and down-regulation of IL-12 p40 secretion by MRL/lprsplenocytes.

The present study also investigated whether TSA and SAHA down-regulateexpression of IL-6 mRNA and protein. It is known in the art that IL-6 isalso a Th2-derived proinflammatory cytokine that promotes B cell growthand differentiation. In SLE, there are increased numbers of circulatingIL-6-producing cells that have increased levels of IL-6 transcript andintracellular IL-6 protein. In particular, IL-6 promotes MHC classII-restricted help of SLE T cell clones for autologous B cells,promoting production of both polyclonal and anti-self antibodies. Highcytokine levels have been detected in cerebrospinal fluid and correlateclinically with central nervous system (CNS) involvement in lupus.Moreover, markedly increased amounts of IL-6 are secreted in the urineduring lupus nephritis. High circulating levels of IL-6 have also beendetected in the MRL/lpr murine model of SLE. Kiberd, “Interleukin-6receptor blockage ameliorates murine lupus nephritis”, J Am Soc Nephrol4:58 (1993). Treatment of these mice as well as NZB/W mice withanti-IL-6 mAb diminishes anti-DNA autoantibody levels, delays the onsetof proteinuria and significantly prolongs longevity. A diminution ofanti-dsDNA autoantibody production is also seen when human SLE B cellsare treated with anti-IL-6 mAb in vitro.

In determining whether TSA or SAHA down-regulate IL-6 mRNA content,splenocytes from 24-wk MRL/lpr mice were treated with increasingconcentrations of TSA for 18 hr. In agreement with previous studiesperformed by the present application, significantly increased,constitutive IL-6 transcripts in 24-wk old mice (FIG. 10A, lane 1) werecompared to 10-wk old mice (FIG. 10B, lane 1). As shown in FIG. 10A, TSAdown-regulated IL-6 mRNA in a dose-dependent manner. TSA decreased IL-6transcript at a concentration of 100 ng/ml and exerted maximalinhibition at 300 ng/ml. To address whether TSA and SAHA down-regulateLPS- and IFN-γ induced IL-6 transcript levels, splenocytes from youngerMRL/lpr mice were treated in the absence or presence of TSA or SAHA for18 hr before LPS and IFN-γ stimulation for 6 or 18 hr. As depicted inFIGS. 10B & 10C, IL-6 mRNA was up-regulated by LPS and IFN-γ stimulation18-fold at 6 hr and 10-fold at 18 hr relative to GAPDH. In contrast,there was no detectable IL-6 mRNA in LPS- and IFN-γ stimulatedsplenocytes following pretreatment with TSA or SAHA (FIG. 10B, lanes4-7). Next, the effect of TSA and SAHA on IL-6 protein secretion instimulated splenocytes was measured (FIG. 10D). When splenocytes werecultured with LPS and IFN-γ for 72 hr, there was a mean (±SEM) 34.4±7.6pg/ml of IL-6 protein secretion. In contrast, treatment with TSA or SAHAin the presence of LPS and IFN-γ for 72hr yielded no detectable IL-6protein secretion (p=0.002, ANOVA FIG. 10D). Taken together, theseresults demonstrated that both TSA and SAHA down-regulated IL-6 mRNA andIL-6 secretion in MRL/lpr splenocytes.

TSA and SAHA Decrease Expression of IL-10 mRNA and Protein

IL-10 is a potent, Th2 growth and differentiation factor for activated Bcells. Current evidence indicates that this cytokine plays a centralrole in autoantibody production. In vivo administration of rIL-10accelerates autoimmunity whereas anti-IL-10 mAb delays the onset ofanti-dsDNA autoantibody production, proteinuria, GN, and decreasesmortality in NZB/W F₁ mice.

In investigating whether TSA down-regulates IL-10 mRNA, MRL/lprsplenocytes were treated with increasing concentrations of TSA. Theheightened, constitutive expression of IL-10 mRNA confirms previousfindings (FIG. 11A & 11B, lane 1). TSA down-regulated IL-10 mRNA at adose of 300 ng/ml. To address whether TSA and SAHA down-regulate LPS-and IFN-γ induced IL-10 transcript, splenocytes from 10-wk old MRL/lprmice were treated in the absence or presence of TSA or SAHA for 18 hrbefore LPS and IFN-γ. stimulation for 6 or 18 hr. FIGS. 11B & 11Cdemonstrate that IL-10 mRNA was up-regulated by LPS and IFN-γstimulation approximately 5-fold at 6hr and 3-fold at 18hr relative toGAPDH transcript. There was no detectable IL-10 mRNA in TSA- orSAHA-pretreated splenocytes when stimulated with LPS and IFN-γ (FIG.11B, lanes 4 - 7).

To determine the effect of TSA and SAHA on IL-10 protein secretion,MRL/lpr splenocytes were stimulated with Con A, LPS, or LPS +IFN-γ for72 hr in the absence or presence of TSA or SAHA. As shown in the FIG.11D, splenocytes stimulated with Con A, LPS, or LPS +IFN-γ secreted amean (±SEM) of 31.8±4.5, 32.3+1.9, and 34.8±4.9 pg/ml, respectively, ofIL-10 protein. When cells were pretreated with TSA or SAHA, there was nodetectable IL-10 secretion when stimulated by either LPS, Con A, or LPS+IFN-γ (For SAHA: p<0.001, p=0.002, p=0.002, respectively; for TSA:p<0.001, p=0.002, p=0.003, respectively, ANOVA). Taken together, theseresults reveal that both TSA and SAHA down-regulate IL-10 mRNA levelsand protein secretion.

TSA and SAHA Induce Accumulation of Acetylated Histones

One mechanism by which HDIs suppress transcription of cytokine genes inMRL/lpr splenocytes may be by increasing accumulation of acetylatedhistones which may result in chromatin remodeling. The accumulation ofacetylated histones H3 and H4 in HDI-treated splenocytes was quantifiedto test this hypothesis. FIG. 12 is an immunoblot analysis of theacetylation levels of H3 and H4 histones. Following exposure of cells toTSA or SAHA for 18 hr, a marked increase in the accumulation ofacetylated H3 and H4 histones was observed (compare Lanes 2-5 to Lane1). These findings suggest that inhibition of HDAC by TSA and SAHApromotes acetylation of H3 and H4 histones, and supports the hypothesisthat this mechanism may be involved in the down-regulation of severalcytokine genes in MRL/lpr splenocytes.

TSA and SAHA Decrease Inducible Nitric Oxide (iNOS) in Splenocytes andNitric Oxide Secretion in Mesangial Cells

Excessive production of nitric oxide (NO) is crucial to the initiationand maintenance of glomerulonephritis in MRL/lpr mice. Pharmacologicinhibition of NO synthesis in MRL/lpr mice abrogates diseaseprogression. In examining whether HDIs could suppress inducible nitricoxide synthetase (iNOS), splenocytes from 10 wk old mice were stimulatedin the presence or absence of TSA or SAHA with LPS and IFN-γ for 24 hr.Similar to Th1 and Th2 cytokines, these HDIs were able to suppress iNOSmRNA induction (FIG. 13A). To determine whether HDIs could suppressnitric oxide secretion by mesangial cells, mesangial cells werestimulated with LPS (1 mg/ml) in the presence of vehicle or TSA for 24hr. The supernatant from the cell culture was assayed for nitric oxide.TSA was able to suppress LPS-induced nitric oxide production bymesangial cells (FIG. 13B).

EXAMPLE 7 In Vivo Study

In Vivo Administration of TSA Decreases Proteinuria,Lymphoproliferation, IFN-γ Secretion, and Physical Symptoms of Lupus inMRL/lpr Mice

TSA was administered to MRL/lpr mice at 12 wks of age coinciding withthe time of onset of clinical disease. TSA was given subcutaneously (sc)at a dose of 0.5mg/kg dissolved in 40 μl DMSO; control mice were giventhe vehicle alone (40 μl DMSO). Proteinuria was assessed as a measure ofalbumin in mg/mouse/day prior to the initial dose (at 12 wks of age) andafter four wks of treatment (at 16 wks of age). The results shown inFIG. 14B demonstrate that TSA prevented an increase in proteinuriaobserved in vehicle treated mice. After four weeks of treatment, ninemice in each group were sacrificed. At necropsy, the weight of thespleens were measured and sera was collected. The spleens weresignificantly decreased in weight in TSA treated mice compared tovehicle controls (FIG. 14A). This suggests that TSA prevents thelymphoproliferation and enlargement of spleens observed as diseaseprogresses in MRL/lpr mice (as seen in vehicle treated mice). IFN-γ wasmeasured by ellispot assay in splenocytes isolated from vehicle or TSAtreated mice. There is a significant decrement in the number of IFN-γsecreting cells in TSA treated mice. In addition, the TSA treated miceshowed signs of better overall health compared to vehicle controls.

In Vivo Administration of TSA Decreases Glomerulonephritis and RenalScore

TSA was administered to MRL/lpr mice at 12 wks of age at the time ofonset of clinical disease. TSA was given subcutaneously (sc) at a doseof 0.5 mg/kg dissolved in 40 μl DMSO; control mice were given thevehicle alone (40 μl DMSO). After four weeks of treatment, nine mice ineach group were sacrificed. One-half of one kidney was fixed in 10%formaldehyde/PBS, embedded in paraffin, and stained with hematoxylin andeosin. An examiner blinded to the treatment group quantified diseaseactivity as follows: (a) Glomeruli were graded for hypercellularity(0-4), crescent formation (0-4), and inflammation (0-4). (b) Theinterstitium was graded for inflammation (0-4). Scores were additive(BALB/c mice historically receive scores of 0-0.5). Half of the secondkidney was snap-frozen, embedded in OCT, frozen sections cut andanalyzed for C3 deposition by immunofluorescence. TSA showed asignificant reduction of glomerulonephritis and renal score (FIG. 15)after 4 weeks treatment, but did not show change in C3 deposition.

In Vivo Administration of TSA does not Decrease Autoantibody Production

In determining whether TSA suppresses autoantibody production anti-dsDNAand anti GBM-auto-antibodies were measured. There was no significantchange between TSA or vehicle treated mice (FIG. 16). This result mayindicate that TSA does not decrease anti-dsDNA levels in mice that havealready developed the antibodies or this time point was too soon to seea positive effect (only 4 weeks treatment).

EXAMPLE 8 Suppression of Proteinuria and Nitric Oxide Secretion by HDIsin MRL/lpr/lpr Mice In Vivo

The effect of TSA on proteinuria and nitric oxide secretion andincreased survival in 26 week old MRL/lpr mice was examined. In this agegroup, MRL/lpr mice developed full-blown disease with evidence ofproliferative immune-complex-mediated glomerulo-nephritis, vasculitis,arthritis, and massive lymphadenopathy. Most of the mice expired by theage of 30 weeks. Twelve 26 week old MRL/lpr mice were randomly selectedto receive 5 mg/kg TSA in 50 ul DMSO or 50 ul DMSO as vehicle control bydaily subcutaneous (s.c.) injection over the course of 4 weeks or untilthey exhibited distress. The data for the first two weeks is shownbelow. Urine was collected for a 24 hr time period on days 0, 7 and 14,and measured for protein and nitric oxide. Toxicity of TSA is beingassessed by monitoring body weight, infection, abnormal behavior andsurvival of treated mice. In comparison to vehicle treated mice, six TSAtreated mice have less proteinuria in day 7 and day 14 compared to day 0(Table 1 and 2). TABLE 1 Proteinuria (ug/day) in vehicle treated miceMOUSE DAY 0 DAY 7 DAY 14 1 41.2 32.7 40.1 2 8.1 4.27 3.15 3 2145 18693511 4 69.5 34.9 65.1 5 533 47 685 6 12.6 4.01 4.76

TABLE 2 Proteinuria (ug/day) in TSA treated mice. MOUSE DAY 0 DAY 7 DAY14 1 16689 274 865 2 213 54 82 3 14420 4701 3081 4 107 80 25 5 5 9 10 6964 340 242 7 10 4.6 4.3

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of treating an autoimmune disease in a subject in needthereof, the method comprising administering to said subject atherapeutically effective amount of suberoylanilide hydroxamic acid or apharmaceutically acceptable salt thereof.
 2. A method of treatingsystemic lupus erythematosus in a subject in need thereof, the methodcomprising administering to said subject a therapeutically effectiveamount of suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt thereof.
 3. A method of treating an autoimmune diseasein a subject in need thereof, the method comprising administering to thesubject a pharmaceutical formulation comprising a pharmaceuticallyacceptable carrier and suberoylanilide hydroxamic acid or apharmaceutically acceptable salt thereof in an amount sufficient totreat a symptom of autoimmune disease.
 4. A method of treating systemiclupus erythematosus in a subject in need thereof, the method comprisingadministering to said subject a pharmaceutical formulation comprising apharmaceutically acceptable carrier and suberoylanilide hydroxamic acidor a pharmaceutically acceptable salt thereof in an amount sufficient totreat a symptom of systemic lupus erythematosus.